Regional diversity in the murine cortical vascular network is revealed by synchrotron X-ray tomography and is amplified with age

Cortical bone is permeated by a system of pores, occupied by the blood supply and osteocytes. With ageing, bone mass reduction and disruption of the microstructure are associated with reduced vascular supply. Insight into the regulation of the blood supply to the bone could enhance the understanding of bone strength determinants and fracture healing. Using synchrotron radiation-based computed tomography, the distribution of vascular canals and osteocyte lacunae was assessed in murine cortical bone and the influence of age on these parameters was investigated. The tibiofibular junction from 15-week- and 10-month-old female C57BL/6J mice were imaged post-mortem. Vascular canals and three-dimensional spatial relationships between osteocyte lacunae and bone surfaces were computed for both age groups. At 15 weeks, the posterior region of the tibiofibular junction had a higher vascular canal volume density than the anterior, lateral and medial regions. Intracortical vascular networks in anterior and posterior regions were also different, with connectedness in the posterior higher than the anterior at 15 weeks. By 10 months, cortices were thinner, with cortical area fraction and vascular density reduced, but only in the posterior cortex. This provided the first evidence of age-related effects on murine bone porosity due to the location of the intracortical vasculature. Targeting the vasculature to modulate bone porosity could provide an effective way to treat degenerative bone diseases, such as osteoporosis

Damage mechanics of human cortical bone

Skeletal fragility is an important orthopedic concern including the prevention of osteoporosis, long-term stability of prosthetic implants and stress fractures. Damage in human cortical bone has been implicated as a cause of increased fragility and is thought to initiate bone remodeling. Therefore, characterization of the mechanisms of damage initiation and accumulation in bone is challenging not only from the engineering prospective but also has a potential of revealing new insights in its physiology. The main objectives of this dissertation work were to study the early stages of damage development in human cortical bone and develop a constitutive formulation describing damage behavior.;Laser Scanning Confocal Microscopy was utilized to study damage genesis. It was found that confocal microscopy allowed detection of the early stages of damage development within the lacunae-canalicular network of cortical bone. Based on those observations and a common knowledge about the operation of mechanosensing cells housed within the tissue it was proposed that damage could initiate bone remodeling much earlier than it is currently believed.;In the second portion of this work the evolutionary properties of chosen damage parameters were investigated under 3-point bending loading. The parameters of interest were the stiffness drop and the permanent strain. It was found that shear stresses play profound role in the failure behavior. It was also shown that damage parameter defined as the stiffness drop after successive cycles is a quadratic function of nonlinear strain. In addition a linear relationship was obtained between the permanent and total strain.;The third portion of this work was concerned with the development and validation of a constitutive model for cortical bone based on the continuum damage mechanics. It was demonstrated that bone is a linear viscoelastic material for stress levels below a threshold value. Beyond the threshold it behaves as a viscoelastic damaging material. Subsequently a coupled viscoelasticity-damage formulation was adopted and a model was derived based on thermodynamics of irreversible processes. The model was simplified for one-dimensional uniaxial case and experiments were performed for the model verification

A New Quantitative Method for the Taxonomic Identification of Tetrapods

The rarity of good fossil samples throughout geologic time frequently makes fossil identification difficult. This dissertation presents a new, multivariate, statistically validated method to identify tetrapods based on quantification of the shapes of microstructural features in cortical bone of the postcranial skeleton. The ultimate goal is to reduce the reliance on rare, near-complete fossil skeletons. The method is validated on a set of 15,745 mammalian microstructural features from eleven diverse species. An additional set of 21,122 microstructural features from one species serve to examine microstructural variation within a single skeleton. Microstructural measurements were made on thin-sections using optical microscopy. Initial tests of the method were applied to extant mammalians whose taxonomic affinities were known. Three case studies comparing: 1) the left tibiae from 11 mammals,: 2) the mid-body of each left rib in Odocoileus virginianus, and: 3) five cross-sections from left rib seven of O. virginianus represented tests of inter-taxonomic, intra-skeletal, and intra-bone microstructural variation, respectively. Principal Component Analysis of measurements on the tibiae of 11 mammals was successful in discerning a taxonomic signal in the shape and size characteristics of primary vasculature, secondary osteons, Haversian canals, primary lacunae, and secondary lacunae. No single microstructure or measurement is sufficient to account for taxonomic variation. Rather, size, shape, and orientation of various microstructural features, in combination, define and distinguish the taxa. Soft Independent Modeling of Class Analogy properly reassigned test samples from several taxa. In contrast with the results from the multi-species set, analysis of the intra-skeletal and intra-bone case studies revealed no pattern of microstructural variation. The data suggest that the microstructural variation within a skeleton is small compared to variation between taxa and that intra-skeleton variation will not affect the overall taxonomic designation. All principal component analyses were tested and found to be significant at the 95% confidence level using Multiple Discriminant Analysis. This work establishes a methodology for using bone microstructural features as a means for reconstructing taxonomic identity and supports continued research on this methodology, with the goal of applying it to rare fossil specimens in order to enable a next-generation approach to paleoecological analysis

Dysmorphology And Dysfunction In The Brain And Calvarial Vault Of Nonsyndromic Craniosynostosis

Craniosynostosis is a premature pathologic fusion of one or more sutures in the calvarial vault. The six calvarial sutures are growth sites between adjacent intramembranous bones, which allow for flexibility during passage through the birth canal and accommodation for the growing brain. Premature fusion results in obvious cranial morphologic abnormality and can be associated with elevated intracranial pressure, visual dysfunction, mental retardation and various forms of subtler learning disability. A category of disease called isolated nonsyndromic craniosynostosis (NSC) represents nearly 85% of cases. It results in prototypical skull deformities and has newly-discovered correlations with poor neuropsychologic and visual functioning. Herein we utilize new techniques in magnetic resonance and three-dimensional computed tomographic analysis to explore neural and bony structural foundations to functional deficit. To our knowledge, this is the first report of evidence of microstructural and functional brain abnormalities in sagittal synostosis, and the first characterization of orbital abnormalities from coronal craniosynostosis that may underlie visual abnormalities

EVALUATING DIFFERENTIAL NUCLEAR DNA YIELD RATES AMONG HUMAN BONE TISSUE TYPES: A SYNCHROTRON MICRO-CT APPROACH

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone is suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this research is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types, with a focus on osteocytes and the 3D quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Methods included: (1) quantifying cortical and cancellous bone volume from each bone-sampling site using Computed Tomography (CT), and (2) visualizing and quantifying osteocyte lacunae using synchrotron radiation micro-Computed Tomographic imaging (SR micro-CT). Regions of interest (ROIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren’s (2014) study. Analyses tested the primary hypothesis that the abundance and density of bone’s cellular spaces vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone ROIs containing a higher lacunar abundance and density. The osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from predominantly cancellous bones. It is hypothesized that soft tissue remnants within the medullary cavities of primarily cancellous skeletal elements are driving the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context. The procurement of small, primarily cancellous bones with associated soft tissues should be preferentially sampled, and no longer dismissed as potential DNA sources in favor of cortical bone tissue

Experimental and theoretical analyses of compression induced muscle damage : aetiological factors in pressure ulcers

Pressure ulcers form a major problem in health care. They often occur when patients are bedridden, wheelchair bound or wearing prostheses. The ulcers can be very painful for the patient and often lead to prolonged hospitalization. In addition, the huge costs involved with treatment and prevention put a heavy burden on heath care budgets. Pressure ulcers occur often: between 14% and 33% of the patients in health care institutions develop an ulcer, ranging from discolouration of the skin to severe wounds involving necrosis of epidermis, extending to underlying bone, tendon and joints. It is clear that pressure ulcers are caused by prolonged mechanical loading, applied at the interface between skin and support surfaces. However, the aetiology of pressure ulcers is poorly understood. This forms an important obstacle in decreasing the unacceptably high prevalence figures. It is anticipated that a better understanding of the mechanobiological pathways leading to cell and tissue damage can lead to a breakthrough in reducing pressure ulcer prevalence. In addition, a solid scientific base may establish tools for objective risk assessment and judgement of preventive measures. The present study focuses on deep ulcers that initiate in skeletal muscle tissue, since deep ulcers are more extensive and often difficult to prevent. To obtain insight into the aetiology of these deep ulcers, it is necessary to understand the transfer from externally applied loads at the skin, to the local conditions that the cells experience within the tissue. In addition, the question which local conditions are harmful to the cell needs to be investigated. By combining knowledge on "what a cell feels" with knowledge on potentially harmful conditions, a better judgement of dangerous situations may be achieved. Although several causes of cell damage may play a role in the initiation of pressure ulcers, the present study focussed on the impact of cell deformations. To investigate the hypothesis that prolonged cell deformations lead to cell damage at clinically relevant strains, an experimental model system was developed. A key requirement of this experimental model is the possibility to study the role of cell deformation on cell damage independently of other possible causes of damage. To achieve this, in-vitro engineered muscle tissue constructs were developed. These constructs were compressed using a newly developed compression device. A custom made incubator system was developed to allow monitoring of the constructs for extended periods of time. In addition, a novel assay was developed to determine the viability of the cells during compression. This assay provides quantitative and spatial information on cell damage throughout a construct in a non-invasive manner, making use of fluorescent dyes which are visualized by confocal microscopy. The compression of the engineered muscle tissue constructs indicated that a significant increase in cell death occurs within 1-2 hours and that higher strain levels led to an earlier increase in damage. In addition, it was demonstrated that cell damage was uniformly distributed across the indented area of the construct, without a gradient in percentage dead cells between the centre and periphery of the constructs. The results strongly suggest that prolonged cell deformation was the predominant cause of cell damage in these experiments. This puts a new light on observations in literature which suggested that ischaemia is not the sole determinant for the onset of pressure ulcers. Nevertheless, more experiments are needed to clarify the role of prolonged cell deformations on cell damage. First, it is recommended that the actual local cell deformations are quantified during compression of the constructs. Furthermore, from the present experiments it could not be excluded that the compression of the constructs decreased the permeability of the construct and hence affected cellular metabolism. In future, measuring diffusion pathways of both small molecules and larger vital molecules, may indicate whether this change in permeability is significant. A numerical model was developed to predict local cell deformations, in response to tissue compression. Since the local cell deformations cannot be a-priori determined on the basis of homogenized tissue deformations, a multilevel finite element approach was adopted. In this approach, cell deformations are predicted from detailed nonlinear finite element analyses of the local microstructures of the tissue, which consist of an arrangement of cells embedded in a matrix material. To avoid unacceptably large computational times, the multilevel model was designed to run on a parallel computer system. Application of the multilevel model showed that the heterogeneity of the microstructure of the tissue has a profound impact on local cell deformations, which highly exceeded macroscopic tissue deformations. Moreover, microstructural heterogeneity led to complex cell shapes and caused non-uniform deformations within the cells. To investigate the evolution of compression induced damage in skeletal muscle tissue, the multilevel model was extended with a damage law, which was derived from the in-vitro experiments. With this model, the compression of muscle tissue against a bony prominence was simulated. The percentage of cell damage in the microstructure of the tissue was computed, which could be extrapolated to the bulk tissue level. In the present form, a schematic geometry was considered that intended to elucidate general patterns of tissue damage evolution. The simulations confirmed that it is not feasible to predict the onset of tissue damage on the basis of externally applied loading conditions at the skin surface alone, since these externally applied loads are not indicative of the local mechanical conditions that the cells experience within the tissue. In addition, the simulations showed that it is necessary to consider the local load history of the cells, and the tolerance of the tissue. These findings may explain why a strikingly large variability in load/time threshold values was found in animal studies, which attempted to relate external mechanical to tissue damage, thereby ignoring the local mechanical conditions within the tissue. At present, it is premature to utilize the models presented in this thesis in clinical practice, since the extrapolation towards human patients requires more research. Clearly, further extensions and validation of the numerical model with experimental animal models will be required. This should finally lead to the application in more realistic cases, involving patient data on geometry and tissue properties. Nevertheless, the present models provided an essential step towards evidence based risk assessment and prevention

Steel and bone: Mesoscale modeling and middle-out strategies in physics and biology

Mesoscale modeling is often considered merely as a practical strategy used when information on lower-scale details is lacking, or when there is a need to make models cognitively or computationally tractable. Without dismissing the importance of practical constraints for modeling choices, we argue that mesoscale models should not just be considered as abbreviations or placeholders for more “complete” models. Because many systems exhibit different behaviors at various spatial and temporal scales, bottom-up approaches are almost always doomed to fail. Mesoscale models capture aspects of multi-scale systems that cannot be parameterized by simple averaging of lower-scale details. To understand the behavior of multi-scale systems, it is essential to identify mesoscale parameters that “code for” lower-scale details in a way that relate phenomena intermediate between microscopic and macroscopic features. We illustrate this point using examples of modeling of multi-scale systems in materials science (steel) and biology (bone), where identification of material parameters such as stiffness or strain is a central step. The examples illustrate important aspects of a so-called “middle-out” modeling strategy. Rather than attempting to model the system bottom-up, one starts at intermediate (mesoscopic) scales where systems exhibit behaviors distinct from those at the atomic and continuum scales. One then seeks to upscale and downscale to gain a more complete understanding of the multi-scale systems. The cases highlight how parameterization of lower-scale details not only enables tractable modeling but is also central to understanding functional and organizational features of multi-scale systems